Epigenetic reprogramming, and specifically the resetting of DNA methylation, is a key event during preimplantation development and in the fetal germline. Changes in the maternal diet or exposure to environmental contaminants can disrupt fetal epigenetic reprogramming and lead to pregnancy failure, developmental disorders or adult-onset disease, but the underlying molecular mechanisms remain unknown. It is important to fill this gap in order to develop informed safety standards for maternal diet and exposure to environmental toxins, and understand the epigenetic basis of adult-onset disease. The long-term goal of the investigators' research is to understand the epigenetic regulation of embryonic development. The objective of this application is to define the role of Vitamin C in epigenetic reprogramming in the embryo. Humans rely entirely on their diet for Vitamin C, which is essential for several physiological processes. Recent findings from our lab indicate that Vitamin C has a novel role in the direct regulation of epigenetic reprogramming, and specifically in DNA de-methylation and activation of germline genes in pluripotent stem cells. This proposal will test the innovative hypothesis that Vitamin C is a critical environmental regulator of epigenetic reprogramming in pluripotent stem cells and the fetal germline. The specific aims are: 1) to determine the mechanism of action of Vitamin C in epigenetic reprogramming. The investigators will test the role of Vitamin C as a potential co-factor of Tet enzymes in the generation of 5-hydroxymethyl-Cytosine at the promoters of germline genes; 2) to determine the functional consequences of exposure to Vitamin C in pluripotent stem cells. The role of Vitamin C will be assessed in the regulation of functions specific to pluripotent stem cells in vitro; and 3) to defie the Windows of Susceptibility (WOS) of Vitamin C-mediated epigenetic reprogramming. The investigators will use a validated mouse model of Vitamin C deficiency to determine the WOS to Vitamin C during the stages when epigenetic reprogramming occurs in vivo. This research is expected to provide fundamental new insights into the dietary and environmental modulation of epigenetic reprogramming during gestation, with significant impact in toxicology, maternal-fetal medicine, reproductive biology and cancer. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to the mission of the NIH because it aims to understand how nutrition and exposure to environmental factors affect stem cells in the fetus during pregnancy. This knowledge will inform better safety standards regarding diet and exposure to toxins during gestation, and may lead to improved pregnancy outcomes and reduced incidence of developmental and adult-onset disorders.